Nobuo Machinaga

Discovery of trans-4-[1-[[2,5-Dichloro-4-(1-methyl-3-indolylcarboxamido)phenyl]acetyl]-(4S)-methoxy-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic acid: an orally active, selective very late antigen-4 antagonist.

We have focused on optimization of the inadequate pharmacokinetic profile of trans-4-substituted cyclohexanecarboxylic acid 5, which is commonly observed in many small molecule very late antigen-4 (VLA-4) antagonists. We modified the lipophilic moiety in 5 and found that reducing the polar surface area of this moiety results in improvement of the PK profile. Consequently, our efforts have led to the discovery of trans-4-[1-[[2,5-dichloro-4-(1-methyl-3-indolylcarboxamido)phenyl]acetyl]-(4S)-methoxy-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic acid (14e) with potent activity (IC(50) = 5.4 nM) and significantly improved bioavailability in rats, dogs, and monkeys (100%, 91%, 68%), which demonstrated excellent oral efficacy in murine and guinea pig models of asthma. Based on its overall profile, compound 14e was progressed into clinical trails. In a single ascending-dose phase I clinical study, compound 14e exhibited favorable oral exposure as expected and had no serious adverse events.

Sphingosine 1-phosphate lyase inhibition by 2-acetyl-4-(tetrahydroxybutyl)imidazole (THI) under conditions of vitamin B6 deficiency

Caramel food colorant 2-acetyl-4-(tetrahydroxybutyl)imidazole (THI) causes lymphopenia in animals through sphingosine 1-phosphate lyase (SPL) inhibition. However, this mechanism of action is partly still controversial because THI did not inhibit SPL in vitro either in cell-free or in cell-based systems. It is thought that the in vitro experimental conditions which have been used so far were not suitable for the evaluation of SPL inhibition, especially in case of cell-based experiments. We speculated that the key factor might be the coenzyme pyridoxal 5′-phosphate (PLP), an active form of vitamin B6 (VB6), because media used in cell-based assays usually contain an excess amount of VB6 which leads to the activation of SPL. By the use of VB6-deficient culture medium, we could regulate apo- (without PLP) and holo- (with PLP) SPL enzyme in cultured cells, resulting in the successful detection of SPL inhibition by THI. Although the observed inhibitory effect was not as strong as that of 4-deoxypyridoxine (a VB6 analog SPL inhibitor), these findings may be useful for further understanding the mechanism of action of THI.

A novel and potent VLA-4 antagonist based on trans-4-substituted cyclohexanecarboxylic acid.

During the course of our study, it was revealed that the poor pharmacokinetic properties of a series of benzoic acid derivatives such as 1 should be attributed to the diphenylurea moiety. Thus, we replaced the diphenylurea moiety in 1 with a 2-(2-methylphenylamino)benzoxazole moiety which mimics the diphenylurea structure. However, this modification resulted in a significant decrease (3, IC(50)=19 nM) in VLA-4 inhibitory activity compared to 1 (IC(50)=1.6 nM). To address this discrepancy, we worked on optimization of the carboxylic acid moiety in compound 3. As a result, our efforts have led to the discovery of trans-4-substituted cyclohexanecarboxylic acid derivative 11b (IC(50)=2.8 nM) as a novel and potent VLA-4 antagonist. In addition, compound 11b exhibited favorable pharmacokinetic properties (CL=3.3 ml/min/kg, F=51%) in rats.

Identification of 4-[1-[3-chloro-4-[N’-(5-fluoro-2-methylphenyl)ureido]phenylacetyl]-(4S)-fluoro-(2S)-pyrrolidinylmethoxy]benzoic acid as a potent, orally active VLA-4 antagonist

Optimization of benzoic acid derivatives by introducing substituents into the diphenyl urea moiety led to the identification of compound 20l as a potent VLA-4 antagonist. Compound 20l inhibited eosinophil infiltration into bronchial alveolar lavage fluid in a murine asthma model by oral dosing and its efficacy was comparable to anti-mouse alpha4 antibody (R1-2). Furthermore, this compound significantly blocked bronchial hyper-responsiveness in the model.

A novel, potent, and orally active VLA-4 antagonist with good aqueous solubility: trans-4-[1-[[2-(5-Fluoro-2-methylphenylamino)-7-fluoro-6-benzoxazolyl]acetyl]-(5S)-[methoxy(methyl)amino]methyl-(2S)-pyrrolidinylmethoxy]cyclohexanecarboxylic acid.

We have carried out the optimization of substituents at the C-3 or the C-5 position on the pyrrolidine ring of VLA-4 antagonist 3 with 2-(phenylamino)-7-fluorobenzoxazolyl moiety for the purpose of improving in vivo efficacy while maintaining good aqueous solubility. As a result, we successfully increased in vitro activity in the presence of 3% human serum albumin and achieved an exquisite lipophilic and hydrophilic balance of compounds suitable for oral administrative regimen. The modification resulted in the identification of zwitterionic compound 7n with (5S)-[methoxy(methyl)amino]methylpyrrolidine, which significantly alleviated bronchial hyper-responsiveness to acetylcholine chloride at 12.5mg/kg, p.o. in a murine asthma model and showed favorable aqueous solubility (JP1, 89 μg/mL; JP2, 462 μg/mL). Furthermore, this compound showed good oral bioavailability (F=54%) in monkeys.

A Controlled Release System for Long-Acting Intravitreal Delivery of Small Molecules

Purpose The short half lives of small molecules in the vitreous requires frequent repeated intravitreal injections that are impractical for treatment of chronic eye diseases. We sought to develop a method for increasing the intravitreal half-life of small-molecule drugs. Methods We adapted a technology for controlled release of drugs from macromolecular carriers for use as a long-acting intravitreal delivery system for small molecules. As a prototype, a small molecule complement factor D inhibitor with an intravitreal half-life of 7 hours was covalently attached to a 4-arm PEG40kDa by a self-cleaving β-eliminative linker with a cleavage half-life of approximately 1 week. Results After intravitreal injection in rabbits, the drug was slowly released in the vitreous, and equilibrated with the retina and choroid. The intravitreal half-life of the intact PEG-drug conjugate in the rabbit was 7 days, and that of the released drug was 3.6 days. We simulated the anticipated pharmacokinetics of the delivery system in human vitreous, and estimated that the half-life of a 4-arm PEG40kDa conjugate would be approximately 2 weeks, and that of the released drug would be approximately 5 days. Conclusions We posit that a linker with a cleavage half life of 2 weeks would confer a half life of approximately 7 days to a released small molecule drug in humans, comparable to the half life of approved intravitreal injected macromolecular drugs. Translational Relevance With this technology, a potent small molecule with an appropriate therapeutic window should be administrable by intravitreal injections in the human at once-monthly intervals.

Scintillation Proximity Assay to Detect the Changes in Cellular Dihydrosphingosine 1-Phosphate Levels

Compounds that modulate the activity of sphingosine 1-phosphate (S1P)-metabolizing enzymes are expected to be potential therapeutic agents for various diseases. Investigation of their potencies requires not only cell-free but also cell-based assays in which intracellular accumulation/depletion of S1P could be monitored. However, conventional methods have limitations to their simplicity, mainly due to the necessity of a separation process that separates S1P from its related substances. Here, we describe a method utilizing a scintillation proximity assay (SPA) for semi-quantifying intracellular [3H]-labeled dihydroS1P ([3H]dhS1P), which is also a substrate for S1P-metabolizing enzymes. We found that uncoated yttrium silicate SPA beads could selectively bind to and detect [3H]dhS1P rather than [3H]dihydrosphingosine (the non-phosphorylated form of [3H]dhS1P). Based on this, we developed a novel cell-based assay system which does not require any organic solvent extraction or chromatographic separation, and confirmed its practicality by using siRNA targeting S1P lyase (S1PL) and known S1PL inhibitors as models. Our results demonstrated that this assay is useful for rapid and easy evaluation of S1PL inhibitors, and could be potentially applicable for all compounds that modulate the activity of S1P-metabolizing enzymes.